Jennifer Y. Kong

Ceramide‐1‐phosphate promotes cell survival through activation of the phosphatidylinositol 3‐kinase/protein kinase B pathway

In this report, we show for the first time that ceramide‐1‐phosphate (C1P) stimulates the phosphatidylinositol 3‐kinase (PI3‐K)/protein kinase B (PKB) pathway, which is a major mechanism whereby growth factors promote cell survival. Also, C1P induced IκB phosphorylation, and enhanced the DNA binding activity of the transcription factor NF‐κB. Apoptotic macrophages showed a marked reduction of Bcl‐XL levels, and this was prevented by C1P. These findings suggest that C1P blocks apoptosis, at least in part, by stimulating the PI3‐K/PKB/ NF‐κB pathway and maintaining the production of antiapoptotic Bcl‐XL. Based on these and our previous observations, we propose a working model for C1P in which inhibition of acid sphingomyelinase and the subsequent decrease in ceramide levels would allow cell signaling through stimulation of the PI3‐K/PKB pathway to promote cell survival.

Palmitate-induced apoptosis in cardiomyocytes is mediated through alterations in mitochondria: prevention by cyclosporin A.

Palmitate, a C16 fatty acid found in high concentrations in the blood in acute myocardial infarction, induces apoptotic cell death. To more completely define the nature and mechanism underlying palmitate-induced cell death, cardiomyocytes were cultured from embryonic chick heart and were treated with palmitate. Concentration-dependent loss of cell viability was established by loss of the ability of palmitate-treated cells to exclude propidium iodide (PI), metabolize 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and retain fluorescein diacetate (FDA). Dual staining with PI and FDA and subsequent analysis by FACS established that palmitate-induced cell death was predominantly necrosis whereas apoptosis occurred in 13% of all dead cells. The low proportion of palmitate-induced apoptosis was confirmed by evaluation of the DNA content or PI fluorescent staining of the DNA of permeabilized cardiomyocytes. A critical role for mitochondria in the pathogenesis of palmitate-induced cell death was demonstrated, for the first time, based on palmitate-induced reduction of mitochondrial activity as assessed by the mitochondrial-selective dye chloromethyl-X-Rosamine and the presence of a greater amount of the mitochondrial marker cytochrome C in the cytosol of palmitate-treated cardiomyocytes than in control cells. Further, cyclosporin that inhibits the development of mitochondrial transition pores blocked palmitate-induced alteration in mitochondrial function and palmitate-induced cell death. We further demonstrated the selectivity of cyclosporin A for the prevention of apoptotic cell death in the heart as there was no alteration in necrotic cell death produced by palmitate with cyclosporin pretreatment. Our data demonstrate the nature of palmitate-induced cell death in cardiomyocytes (both apoptotic and necrotic), propose a mitochondrial basis for its pathogenesis and show that cyclosporin A prevents palmitate-induced apoptotic cardiomyocyte cell death.

Ceramide 1-phosphate stimulates macrophage proliferation through activation of the PI3-kinase/PKB, JNK and ERK1/2 pathways

Ceramide 1-phosphate (C1P) was first shown to be mitogenic for fibroblasts, but the mechanisms whereby it stimulated cell proliferation have remained largely unknown. Here we demonstrate that C1P stimulates DNA synthesis and cell division in murine bone marrow-derived macrophages. C1P caused rapid phosphorylation of protein kinase B (PKB, also known as Akt), a downstream target of phosphatidylinositol 3-kinase (PI3-K). Selective inhibition of PI3-K blocked both DNA synthesis and cell growth. C1P induced phosphorylation of GSK-3beta, which is a major target of PKB, and this effect was also abolished by inhibition of PI3-K. In addition, C1P upregulated the expression of cyclin D1 and c-Myc, two major targets of GSK-3beta, which are important regulators of cell proliferation. C1P stimulated the activity of NF-kappaB, and inhibitors of this transcription factor completely blocked macrophage proliferation. Lastly, C1P induced phosphorylation of the mitogen activated protein kinases (MAPK) extracellularly regulated kinases 1 and 2 (ERK1/2), and c-Jun N-terminal kinase (JNK). Inhibition of ERK1/2 and JNK also blocked C1P-induced macrophage proliferation. It can be concluded that C1P stimulates macrophage proliferation through activation of the PI3-K/PKB, ERK and JNK pathways, and that GSK-3beta, c-Myc, cyclin D1, and NF-kappaB are important downstream effectors in this action.

Sphingosine-1-phosphate inhibits acid sphingomyelinase and blocks apoptosis in macrophages

Sphingosine‐1‐phosphate (Sph‐1‐P) regulates critical cellular functions including cell proliferation, differentiation, angiogenesis, and cell survival. However, its mechanisms of action are incompletely understood. Here, we show a novel biological effect of Sph‐1‐P: inhibition of acidic sphingomyelinase (A‐SMase) activity in apoptotic bone marrow‐derived macrophages. A‐SMase catalyzes the conversion of sphingomyelin to ceramides, which are pro‐apoptotic. This action of Sph‐1‐P prevents the accumulation of ceramides and blocks apoptosis, thereby promoting survival of the macrophages.

Ceramide activates a mitochondrial p38 mitogen-activated protein kinase: A potential mechanism for loss of mitochondrial transmembrane potential and apoptosis

This study examined the impact of ceramide, an intracellular mediator of apoptosis, on the mitochondria to test the hypothesis that ceramide utilized p38 MAPK in the mitochondria to alter mitochondrial potential and induce apoptosis. The capacity of ceramide to adversely affect mitochondria was demonstrated by the significant loss of mitochondrial potential (ΔΨm), indicated by a J-aggregate fluorescent probe, after embryonic chick cardiomyocytes were treated with the cell permeable ceramide analogue C2-ceramide. p38 MAPK was identified in the mitochondrial fraction of the cell and p38 MAPK phosphorylation in this mitochondrial fraction of the cell occurred with ceramide treatment. In addition, SAPK phosphorylation and a decrease in ERK phosphorylation occurred in whole cell lysates after ceramide treatment. The p38 MAPK inhibitor SB 202190 but not the MEK inhibitor PD 98059 significantly inhibited ceramide-induced apoptosis and loss of ΔΨm. These data suggest that p38 MAPK is present in the mitochondria and its activation by ceramide indicates local signaling more directly coupled to the mitochondrial pathway in apoptosis. (Mol Cell Biochem 278: 39–51, 2005)

Angiotensin II induces activation of phosphatidylinositol 3-kinase in cardiomyocytes.

Background Phosphatidylinositol 3-kinase phosphorylates membrane lipids at the third position of the inositol ring producing phosphoinositides, not on the pathway for production of 1,4,5-triphosphate. Objective To test the hypotheses that angiotensin II (Ang II) activates phosphatidylinositol 3-kinase in cardiomyocytes and that this pathway is involved in Ang II-induced protein synthesis. Methods Cardiomyocytes, in culture, from 7-day-old chick embryonic hearts were treated with Ang II and the activation of phosphatidylinositol 3-kinase was assessed after immunoprecipitation with antibodies to the p85 subunit of phosphatidylinositol 3-kinase by the conversion of PI (phosphatidylinositol) to phosphatidylinositol 3-monophosphate (PIP) in the presence of g-[32P]-ATP and analyzed by thin-layer chromatography. Western blotting was performed after antiphosphotyrosine immunoprecipitation with antibodies to the p85 subunit of phosphatidylinositol 3-kinase. Protein synthesis was assessed by [35S]-methionine incorporation and polyacrylamide gel electrophoresis. Results Ang II stimulated phosphatidylinositol 3-kinase activity dramatically, with 4.5- and 3.5-fold increases in PIP formation after 1 and 5 min, respectively. The involvement of tyrosine kinases was demonstrated by Western blotting in which Ang II increased tyrosine phosphorylation of a protein recognized by antibodies to the 85 kDa subunit of phosphatidylinositol 3-kinase. Furthermore, the tyrosine kinase inhibitor lavendustin A blocked Ang II-stimulated phosphatidylinositol 3-kinase activity and conversion of phosphatidylinositol to PIP. Ang II increased new protein synthesis as reflected by the significantly (P < 0.05) greater incorporation of [35S]-methionine into cardiomyocytes treated with Ang II. The link between Ang II and protein synthesis was mediated in part through phosphatidylinositol 3-kinase because the phosphatidylinositol 3-kinase inhibitor wortmannin blocked the effect of Ang II on protein synthesis. Increased production both of nuclear and of cytosolic proteins was demonstrated by agarose gel electrophoresis of these cellular components of Ang II-treated cardiomyocytes. Wortmannin produced a general inhibition of the synthesis of nuclear and cytosolic proteins, with a greater effect on nuclear proteins. The action of wortmannin on nuclear protein synthesis was confirmed by similar findings with another phosphatidylinositol 3-kinase inhibitor, LY294002. Conclusion Phosphatidylinositol 3-kinase activation by Ang II occurs through a pathway utilizing tyrosine phosphorylation. Furthermore, this pathway is involved in cardiomyocyte protein synthesis and the possibility that it is operative in Ang II-mediated cardiac hypertrophy arises.

Mitochondrial effects with ceramide-induced cardiac apoptosis are different from those of palmitate

The objective of this study was to evaluate whether ceramide, palmitate, and inhibitors of mitochondrial electron transport chain shared similar effects on the mitochondria of intact cardiomyocytes in order to determine the likelihood that ceramide and palmitate utilize similar mitochondrial mechanisms or pathways to apoptosis. In embryonic chick cardiomyocytes, ceramide, 100 microM for 24h, induced a 42.9+/-5.8% increase in cell death assessed by the MTT assay, and a significant (P<0.01) 3.9+/-0.6-fold increase in apoptosis assessed by propidium iodide staining of permeabilized cells. Mitochondrial potential (delta psi (m)), as demonstrated microscopically and by flow cytometry of cardiomyocytes stained with a J-aggregate dye, was markedly and significantly reduced by ceramide, palmitate, and two different inhibitors of the mitochondrial electron transport chain-rotenone and antimycin A. In contrast, the effect on mitochondria as assessed by CMX-Ros oxidation was dramatically different, as palmitate, rotenone, and antimycin A each produced a reduction, while ceramide increased CMX-Ros fluorescence. Further ceramide-induced cardiomyocyte apoptosis and loss of delta psi (m) operated through a cyclosporine-insensitive pathway similar to rotenone and antimycin A but distinct from palmitate which induced apoptosis though a cyclosporine-sensitive mechanism in these cells. These data suggest that ceramide acts on the mitochondria of intact cells through a cyclosporine-insensitive mechanism likely from a combination of actions including production of mitochondrial oxidants. The discordant findings between ceramide and palmitate suggest that palmitate-induced cell death is not primarily mediated by de novo ceramide synthesis.

Nifedipine does not induce but rather prevents apoptosis in cardiomyocytes

The potential of Ca(2+) channel antagonists, particularly nifedipine, to cause apoptotic cell death has been controversial and is of considerable importance for cardiomyocytes as loss of these cells is an important component of the pathophysiology leading to heart failure. To examine the hypothesis that nifedipine induces cell death and modulates calcium-induced apoptosis, cardiomyocytes in culture from embryonic chick hearts, that readily manifest apoptosis, were studied. Apoptosis was evaluated by fluorescent activated cell sorting (FACS) analysis and by quantitative analysis of DNA fragmentation by an enzyme-linked immunosorbent assay (ELISA) specific for histone-associated DNA fragments of mono- and oligonucleosome size. Cell death was evaluated by using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. Cardiomyocytes were treated with various concentrations of nifedipine over a concentration range relevant to serum concentrations in man. Nifedipine, 1 to 100 microM, did not produce cell death in cardiomyocytes. There was no evidence of apoptosis on FACS analysis of cardiomyocytes stained with fluoresceine diacetate or propidum iodide (PI). Neither was there any evidence of apoptotic nuclei on PI staining of permeabilized cardiomyocytes treated with nifedipine. In contrast, DNA fragmentation consistent with apoptosis was induced in a significant (P<0.05) concentration-dependent manner, by increases in extracellular Ca(2+) concentration ([Ca(2+)](o)). Importantly, nifedipine reduced DNA fragmentation produced by increased [Ca(2+)](o). Furthermore, nifedipine blocked calcium-induced cell death as high [Ca(2+)](o) significantly (P<0. 05) reduced cell viability. These data indicate that nifedipine does not induce apoptosis in cardiomyocytes rather apoptosis in cardiomyocytes is under regulatory control by Ca(2+) and nifedipine can antagonize Ca(2+)-mediated apoptotic cell death.

Angiotensin II does not induce apoptosis but rather prevents apoptosis in cardiomyocytes.

The ability of angiotensin II (ang II) to produce apoptosis is controversial. Cardiomyocytes, isolated from 7-day embryonic chick hearts and maintained in culture for 72 h, were treated with ang II. There was no evidence of ang II-induced apoptosis consistently demonstrated by six different techniques: electrophoretic separation of fragmented DNA, staining with TUNEL, enzyme-linked immunosorbent assay specific for fragmented DNA, dual staining of cells with fluorescein diacetate and propidium iodide with analysis by flow cytometry, staining of nuclei with propidium iodide and cell microscopy. In contrast, apoptosis was readily induced by camptothecin or staurosporine or serum deprivation. The absence of ang II-induced cell death was also demonstrated in neonatal mouse cardiomyocytes in culture. We further sought to answer the question whether ang II Type 1 receptor blockade by antagonizing the potential beneficial effects mediated through this receptor and producing more ang II binding to the ang II Type 2 receptors, would lead to cardiac apoptosis. There was no evidence of ang II-induced apoptosis in the presence of the ang II Type 1 receptor antagonist losartan in embryonic chick cardiomyocytes. Rather ang II prevented serum deprivation-induced apoptosis. In summary, in these cardiomyocytes ang II does not induce but rather prevents apoptosis.

Cytoskeletal actin degradation induced by lovastatin in cardiomyocytes is mediated through caspase-2

The objective of this study was to test the hypothesis that cytoskeletal actin fragmentation is mediated through caspase‐2, specifically examining the ability of a caspase‐2 inhibitor to interfere with actin fragmentation, in comparison with a caspase‐3 inhibitor. Cardiomyocytes were cultured from embryonic chick heart. The fine structural element of cellular F‐actin was visualized by staining cardiomyocytes with NBD‐phallacidin. Lovastatin induced a dramatic and concentration‐dependent loss of intact F‐actin. The selectivity of this effect of lovastatin was demonstrated by the absence of similar changes in F‐actin when cardiomyocytes were treated with the apoptotic stimulus palmitate, the metabolism of which produces acetyl CoA, the early substrate of cholesterol synthesis, through the mevalonate pathway. FACS analysis of NBD‐phallacidin‐stained cells was used to quantify the amount of F‐actin loss. Actin fragmentation produced by lovastatin was operative through a caspase‐2 pathway, as the caspase‐2 inhibitor, z‐VDVAD‐fmk, significantly blocked lovastatin‐induced changes in F‐actin, but the caspase‐3 inhibitor, Ac‐DEVD‐CHO, did not. Interruption of the mevalonate pathway was in part responsible for lovastatins action, as the downstream metabolite mevalonate partially reversed the effect of lovastatin on actin fragmentation. These data indicate a previously unrecognized link between cytoskeletal actin and caspase‐2.